PROJECT SUMMARY/ ABSTRACT Regulation of synapse homeostasis is essential for normal brain development and function, and intimately dependent on astrocytes and microglia -- the support cells of the brain. Astrocytes contact thousands of synapses and promote developmental synapse formation. Microglia are brain-resident immune cells increasingly implicated in both synapse formation and pruning. We have discovered a novel signaling circuit between astrocytes and microglia that promotes synapse elimination in the developing central nervous system (CNS). We found that astrocytes express the immune signal Interleukin-33 (IL-33) whereas microglia express the IL-33 receptor (IL1RL1.) We subsequently showed that in the spinal cord, eliminating IL-33 from developing astrocytes leads to excess synapses, and that IL-33 signals to microglia to drive synapse engulfment and lead to synapse depletion. Our central hypothesis is that astrocytes express and release IL-33 in response to neuron-derived signals, and that IL-33 in turn drives microglial synapse elimination. We will test this hypothesis in three distinct but interrelated aims, focusing on a well-defined and experimentally accessible circuit in the ventrobasal sensory thalamus (VB), where IL-33 is highly expressed during synapse refinement. In Aim One we will determine how astrocytic IL-33 regulates thalamic synapse subtypes and circuit function. We previously showed that global deletion of IL-33 lead to hyperexcitability of the VB circuit and excess synapses. Here we will conditionally delete IL-33 from astrocytes and explore these phenotypes in more detail, quantifying subtypes of afferent excitatory and inhibitory synapses to understand how different components of the circuit are altered. In Aim Two, we will determine the molecular mechanisms regulating microglial synapse engulfment. We previously found that IL-33 promotes engulfment of postsynaptic proteins by microglia. Here, using both standard and high resolution techniques (expansion microscopy), we will quantify engulfment of both pre- and postsynaptic excitatory elements, as well as inhibitory synapses. We will test the requirement for direct signaling to microglia via conditional deletion of its receptor. In Aim 3, we will identify neuronal molecules that induce astrocyte expression and release of IL-33. Our preliminary data demonstrates that norepinephrine is a neuron-derived cue that promotes expression of IL-33 in gray matter astrocytes. Here we will further test which noradrenergic receptors on astrocytes mediate this effect in vivo. We will also test the hypothesis that extracellular release of IL-33 is dependent on neuronal synaptic activity, by modulating activity in vitro and in vivo. Together, these three aims explore the role of a novel glial-neuronal circuit mediating synapse homeostasis. We predict that a broader understanding of how glia communicate via immune molecules to regulate synapses will fundamentally impact our understanding of how neural circuits change in learning and development as well as in neurodevelopmental diseases.